HK1059379A - Gene detection assay for improving the likelihood of an effective response to an erbb antagonist cancer therapy - Google Patents

Description

Gene detection assays for increasing the likelihood of an effective response to ErbB antagonist cancer therapy

This application claims priority to provisional application 60/205,754 filed on 19/5/2000 as per the provisions of 35 u.s.c.119(e), which application is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to the treatment of cancer characterized by the overexpression of a tumor antigen, such as an ErbB receptor, in particular HER 2. More particularly, the invention relates to more effective treatment of a patient susceptible to cancer or a patient having cancer whose tumor cells have been determined to overexpress ErbB by a gene amplification assay with an ErbB antagonist, such as an anti-ErbB antibody. The invention also provides a pharmaceutical pack for such treatment.

Technical Field

Further knowledge of the technology and epidemiology of genetics and development has made it possible to correlate genetic abnormalities with certain malignant diseases and the assessment of an individual's risk of developing a particular malignant disease. However, most of the existing methods for assessing the risk of or predisposition to develop malignant disease in a tissue associated with an individual have well-known disadvantages. For example, methods that require tissue disaggregation, such as Southern, Northern, or Western blot analysis, are less accurate due to the mixing of malignant cells with normal or non-malignant cells from the same tissue. Furthermore, disruption of tissue architecture results in the inability to link malignant cells to the presence of genetic abnormalities from a morphological specificity standpoint. This is particularly problematic in tissue types known to be heterogeneous, such as human breast cancer, where there may be a large number of non-malignant cells in any one region.

The HER2/neu gene encodes a protein product, commonly referred to as p185HER 2. The native p185HER2 protein is a membrane receptor-like molecule with homology to the Epidermal Growth Factor Receptor (EGFR). Amplification and overexpression of HER2 in human breast Cancer has been associated in some studies with short disease-free intervals and short overall survival (van de Vijer et al New Eng. J. Med. 317: 1239 (1988); Walker et al Br. J. Cancer 60: 426 (1989); Tandon et al J. Clin. invest.7: 1120 (1989); Wright et al Cancer Res.49: 2087 (1989); McCann et al Cancer Res 51: 3296 1991; Paterson et al Cancer Res.51: 556 (1991); and Winstanleiyetal. Br. J. Cancer 63: 447 (1991); Eucalyptus et al Oncogene 4: 105 (1989); Hench et al Lab 1990: 160: 1990: 26: J.) while others have not been described (Zhou et al Oncogene 4: 105: 1989; Oc Pan et al 1989); Octaz et al Cancer 160: 1990: 26: J.944).

In an early evaluation of 103 breast cancer patients, those with more than three tumor cell positive axillary lymph nodes (node positive) had a greater propensity to overexpress the HER2 protein than those with fewer than three positive lymph nodes (Slamon et al Science 235: 177 (1987)). In subsequent evaluations of 86 node-positive breast cancer patients, there was a significant correlation between gene amplification, early recurrence, and short survival. Overexpression of HER2 can be determined by Southern and Northern blotting and correlates with the expression of HER2 oncoprotein as assessed by Western blotting and Immunohistochemistry (IHC) (Slamon et al Science 235: 177 (1987); Slamon et al Science 244: 707 (1989)). As a result, the survival rate of patients carrying more than 5 copies of her2 gene was found to be shortened by nearly 5 times in the middle stage compared with those without gene amplification. This correlation persists even after correcting nodal status and other prognostic factors in multivariate analysis. These studies were also performed in 187 additional node-positive patients and showed that gene amplification, increased amounts of mRNA (as detected by Northern blotting), and increased expression of protein (as detected by immunohistochemical methods) were also associated with shortened survival (Slamon et al Science 244: 707 (1989)); (see also U.S. Pat. No. 4,968,603). Nelson et al compared her2/neu gene amplification using FISH and immunohistochemistry (Nelson et al Modern Pathology 9(1)21A (1996)).

Immunohistochemical staining of tissue sections is a reliable method to assess protein changes in heterogeneous tissues. Immunohistochemistry (IHC) technology utilizes antibodies to detect and observe antigens of cells in situ, mainly by chromogenic reactions or fluorescent methods. This technique has advantages by avoiding the unexpected consequences of tissue disaggregation and allowing single cells to be evaluated morphologically. Furthermore, the target protein is not altered during freezing.

However, IHC performed on formaldehyde fixed and paraffin embedded tissue samples in Clinical Trial Analysis (CTA) showed only 50% -80% sensitivity relative to frozen IHC samples (Press, cancer research 54: 2771 (1994)). Thus, IHC can lead to false negative results, leaving some patients who can benefit from treatment excluded from treatment.

Fluorescence In Situ Hybridization (FISH) is a recently developed method to directly assess the presence of genes in intact cells. FISH is an attractive method to assess the presence of malignant states in paraffin-embedded tissues because it is cell-specific and overcomes the problem of cross-linking and other effects of protein changes caused by formalin fixation. FISH has been used in conjunction with classical staining methods to find associations between genetic abnormalities and cell morphology (see, e.g., Anastasi et al, Blood 77: 2456-.

To date, HER2 gene amplification was not found to be associated with the outcome of anti-HER 2 antibody treatment, only with disease prognosis. The standard assay is IHC on formalin fixed and paraffin embedded samples. When these samples scored 3+ or 2+, the patient was identified as one likely to benefit from treatment with an anti-HER 2 antibody (such as Herceptin ). Scores of 3+ and 2+ are associated with her2 gene amplification and can be verified, for example, by FISH. However, there is still a need for more effective identification of candidates for successful treatment with ErbB antagonists, such as successful treatment with Herceptin .

Disclosure of Invention

The present invention advantageously provides a method of increasing the likelihood of effectiveness of an ErbB antagonist cancer therapy. The method comprises administering to a subject in which the ErbB gene in tumor cells of a tissue sample is amplified a cancer treating dose of an ErbB antagonist. Preferably ErbB is HER 2. In a particular embodiment, the method further comprises administering a cancer treating dose of a chemotherapeutic drug, particularly paclitaxel (taxol).

In a particularly preferred embodiment as exemplified herein, the invention provides a method of increasing the likelihood of effectiveness of an anti-HER 2 antibody in treating cancer. The method comprises administering a cancer treating dose of an anti-HER 2 antibody to a subject who has had HER2 gene amplified in tumor cells of a tissue sample from the subject.

The present invention demonstrates that gene amplification is more effective in indicating antibody-based tumor therapy than protein detection by immunohistochemistry, an unexpected clinical outcome that has led to an overall expansion of tumor antigens. Thus, any anti-tumor specific antigen-based antibody therapy can increase the likelihood that a patient whose gene encoding the tumor antigen has been genetically amplified will be successfully treated.

The invention is particularly advantageous in that it allows the selection of patients in need of treatment not according to immunohistochemical criteria. Thus, in a specific embodiment, a formaldehyde-fixed tissue sample from a subject is immunohistochemically assigned an antigen level score of 0 or 1 +.

The invention also provides a pharmaceutical pack comprising an ErbB antagonist for use in the treatment of cancer and instructions for administering the ErbB antagonist to a patient whose tumor cells in a tissue sample have been found to have amplification of the ErbB gene. Preferably the ErbB antagonist is an anti-ErbB antibody, such as an anti-HER 2 antibody. In another aspect, the above description also indicates the administration of a cancer treating dose of a chemotherapeutic agent, such as paclitaxel. Such a kit may be provided for any antibody-based therapy specific for a tumor-specific antigen, including instructions for use.

Detailed Description

The present invention is advantageous in that patients who are more likely to respond to such administration, who are found to have amplified genes encoding such tumor antigens or ErbB receptor proteins, can be effectively treated by administering a therapeutic drug, i.e., a therapeutic antibody against a tumor antigen or an ErbB receptor antagonist. The present invention is based, in part, on the following unexpected findings: HER2 gene amplification, detected by, for example, Fluorescence In Situ Hybridization (FISH), although correlated with HER2 expression detected by Immunohistochemistry (IHC), more accurately selected patients receiving treatment because FISH status was unexpectedly found to be more correlated with response to treatment. This result is in a sense unexpected because FISH correlates with a Clinical Trial Analysis (CTA) IHC assay to the same extent as another IHC assay (HercepTest). Based on this finding, FISH is expected to have a similar correlation with responsiveness to treatment. Another reason for this surprising result is that direct assays for proteins (by immunoassay) are expected to more accurately assess cancer therapy targeting the protein than indirect assays for expression (e.g., gene amplification).

Evaluation of different groups and subgroups of patients confirmed that gene amplification analysis enabled selection of those patients who are likely to respond to treatment. IHC can be used to score HER2 expression on tumor cells: 0 (no expression) to 3+ (very high level expression). Clinical selection criteria excluded patients scored 0 and 1+ and selected patients scored 2+ and 3 +. The data show that 14% of patients scored 2+/3+ responded to Herceptin  and 20% of patients with FISH + (amplified her2 gene) responded to Herceptin . The subgroup scored as 3+ had a response rate of 17%, which is very close to the response rate of FISH + patients. However, the subgroup scored 2+ showed less than half of the response rate of FISH + patients. Thus, gene amplification clearly distinguishes the major subset in the 2+ subset, allowing more effective treatment of those with FISH +, and quickly identifying patients who are eligible for and in urgent need of other forms of treatment.

Gene amplification analysis also identifies patients who were not necessarily excluded but were excluded due to abnormalities in IHC analysis, particularly when tested on formalin-fixed and paraffin-embedded samples (which may destroy the antibody epitope on HER2 protein, but have much less impact on gene amplification analysis). As described in the examples, one group of patients with scores of 0 and 1+ is FISH +. These patients tend to respond to anti-HER 2 antibody treatment, e.g. with Herceptin , but according to IHC criteria they will be excluded from receiving the treatment.

Thus, the present invention is advantageous in that patients who are likely to benefit from treatment, but who are excluded from treatment as judged by conventional IHC criteria, can be included in the treatment scope. At the same time, the present invention also enables the exclusion of those patients who should rapidly seek other modes of treatment because anti-tumor antigen therapy (i.e., ErbB antagonists or tumor-specific therapeutic antibodies) is unlikely to be successful.

Briefly, the present invention is an effective adjunct to IHC assays in selecting patients based on the expression level of a target protein. It also enables preliminary screening and selection of patients alone, i.e. without IHC. The present invention significantly improves the screening and selection of patients receiving anti-tumor antigen therapeutic antibody therapy, ErbB receptor antagonist therapy, and other therapies targeting over-expressed tumor antigens (or tumor-specific antigens) at cancer therapeutic doses, thereby increasing the likelihood that these patients will benefit from such therapies.

Another aspect of the invention relates to an article of manufacture or package of manufacture (package) comprising a container and optionally a label on the container and a package insert, the container containing a composition comprising an ErbB antagonist, such as an anti-ErbB antibody (or other anti-tumor-specific antigen antibody), said label indicating that said composition can be used in the treatment of conditions in which an ErbB receptor is overexpressed, said package insert containing instructions for administering said antagonist to a patient who has been found to have an amplified ErbB gene.

Definition of

Herein, the "ErbB receptor" is a receptor protein tyrosine kinase belonging to the ErbB receptor family, including the EGFR, HER2, ErbB3 and ErbB4 receptors, as well as TEGFR (us patent 5,708,156) and other members of this family identified in the future. ErbB receptors typically include an extracellular domain that binds ErbB ligands; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxy-terminal signal domain containing several tyrosine residues that can be phosphorylated. The ErbB receptor may be a native sequence ErbB receptor or an amino acid sequence variant thereof. Preferably the ErbB receptor is a native sequence human ErbB receptor.

The ErbB receptor is a tumor-specific antigen or one of the tumor antigens. The term "tumor antigen" as used herein refers to a protein that is expressed at a higher level in tumor cells than in normal cells. Normal cells typically used for comparison are cells of the same tissue type (particularly phenotype) as the tumor, or as the original tissue cells from which the tumor originated. By "tumor-specific antigen" is meant an antigen that is expressed predominantly on tumor cells or only on tumor cells. Examples of tumor-specific antigens include, in addition to the ErbB receptor, MART1/MelanA, gp-100, and tyrosinase (in melanoma); MAGE-1 and MAGE-3 (in bladder, head and neck, non-small cell carcinoma); HPV EG and E7 proteins (in cervical cancer); mucin/MUC-1 (in breast, pancreatic, colon and prostate cancers); prostate specific antigen/PSA (in prostate cancer); and carcinoembryonic antigen/CEA (in colon, breast and gastrointestinal cancers).

By "amplified" is meant that there are one or more additional copies of erbB or other gene encoding a tumor antigen in a set of chromosomes. Gene amplification can lead to overexpression of proteins such as ErbB receptor proteins. Gene amplification in cells from a tissue sample can be determined by a variety of techniques, particularly Fluorescence In Situ Hybridization (FISH), but also includes, but is not limited to, quantitative PCR, quantitative Southern hybridization, and the like.

By "tissue sample" is meant a collection of similar cells obtained from a subject or patient tissue, preferably including nucleated cells with chromosomal material. The 4 major human tissues are: (1) epithelial tissue; (2) connective tissue, including blood vessels, bone and cartilage; (3) muscle tissue; and (4) neural tissue. The source of the tissue sample may be a solid tissue, such as a fresh, cryogenically and/or preserved organ or tissue sample or biopsy sample or aspirate; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid, intraperitoneal body fluid, or interstitial fluid; cells from any stage of pregnancy or development in a subject. The tissue sample may also be primary cells or cultured cells or cell lines. The tissue sample may contain compounds that are not naturally mixed with the tissue, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. In one embodiment of the invention, the tissue sample is a "non-hematologic tissue" (i.e., not blood or bone marrow tissue).

A "slice" of a tissue sample herein refers to a single portion or piece of the tissue sample, e.g., a slice of tissue or cells cut from the tissue sample. It is to be understood that multiple sections of a tissue sample may be taken and analyzed in accordance with the present invention, provided that the present invention encompasses methods that allow both morphological and molecular level analysis, or both protein and nucleic acid analysis of the same section of a tissue sample.

By "correlating" is meant that the performance and/or results of a first analysis are related to the performance and/or results of a second analysis in any way. For example, the results of the first analysis may be used to perform the second analysis, and/or the results of the first analysis may be used to determine whether the second analysis is required, and/or the results of the first analysis may be compared to the results of the second analysis. In the IHC versus FISH relationship, IHC results can be used to determine whether FISH is required and/or to compare protein expression levels to gene amplification, thereby further identifying tumor biopsy specimens (e.g., comparing HER2 protein expression to HER2 gene amplification). One advantage of the present invention is that it enables the identification of patients who IHC show low levels of their antigen, but who are likely to benefit from treatment as judged by FISH.

"nucleic acid" includes any DNA or RNA present in a tissue sample, e.g., of chromosomal, mitochondrial, viral and/or bacterial origin. The term "nucleic acid" includes one or both strands of a double-stranded nucleic acid molecule, and also includes any fragment or portion of an entire nucleic acid molecule.

"Gene" refers to any nucleic acid sequence or portion thereof that encodes or transcribes RNA (rRNA, tRNA, or mRNA, which is translated into protein) or regulates the expression of another gene. A gene may consist of all the nucleic acids responsible for encoding a functional protein, or only the part of the nucleic acids responsible for encoding or expressing a protein. The nucleic acid sequence may contain a genetic abnormality in an exon, an intron, an initiation or termination region, a promoter sequence, other regulatory sequences, or a unique region contiguous with the gene.

An "ErbB ligand" refers to a polypeptide that binds to and/or activates an ErbB receptor. ErbB ligands of particular interest herein are native sequence human ErbB ligands, such as Epidermal Growth Factor (EGF) (Savage et al, J.Bio.chem.247: 7612-7621 (1972)); transforming growth factor alpha (TGF-. alpha.) (Marquardt et al, Science 223: 1079-1082 (1984)); diregulin, also known as Schwannoma cell (schwanoma) or keratinocyte autocrine growth factor (Shoyab et al Science 243: 1074-1076 (1989); Kimura et al Nature 348: 257-260 (1990); and Cook et al mol.cell.biol.11: 2547-2557 (1991)); beta cell regulators (Shing et al, Science 259: 1604-1607 (1993); Sasada et al biochem. Biophys. Res. Commun.190: 1173 (1993)); heparin-binding epidermal growth factor (HB-EGF) (Higashiyama et al, Science 251: 936-939 (1991)); epidermal regulators (Toyoda et al, J.biol.chem.270: 7495-7500 (1995); and Komurasaki et al Oncogene 15: 2841-2848(1997)), genetic regulatory proteins (described below); neuregulin-2 (NRG-2) (Carraway et al, Nature 387: 512-516 (1997)); neuregulin-3 (NRG-3) (Zhang et al, Proc. Natl. Acad. Sci.94: 9562-9567 (1997)); or cripto (CR-1) (Kannan et al J.biol. chem.272 (6): 3330-3335 (1997)). ErbB ligands that bind EGFR include EGF, TGF- α, amphiregulin, β -cell regulator, HB-EGF and epidermal regulator. ErbB ligands that bind HER3 include genetic regulatory proteins. ErbB ligands capable of binding HER4 include betaregulators, epiregulins, HB-EGF, NRG-2, NRG-3 and genetic regulatory proteins.

Herein, "genetic regulatory protein (Heregulin)" refers to a protein as described in us patent 5,641,869 or marchinoni et al, Nature, 362: 312-318(1993), polypeptides comprising an amino acid sequence encoded by a gene product of a genetic regulatory protein and biologically active variants of said polypeptides. Examples of genetic regulatory proteins include genetic regulatory protein-alpha, genetic regulatory protein-beta 1, genetic regulatory protein-beta 2 and genetic regulatory protein-beta 3(Holmes et al, Science, 256: 1205-1210 (1992); and U.S. Pat. No. 5,641,869); neu Differentiation Factor (NDF) (Peles et al Cell 69: 205-216 (1992)); acetylcholine receptor-inducing activity (ARIA) (cells 72: 801-815(1993)) in Falls et al; glial Growth Factor (GGF) (Marchionni et al, Nature, 362: 312-318 (1993)); sensory and motor neuron derived factor (SMDF) (Ho et al J.biol.chem.270: 14523-; gamma-genetic regulatory protein (Oncogene 15: 1385-1394(1997)) by Schaefer et al. An example of a biologically active fragment/amino acid sequence variant of a native sequence HRG polypeptide is an EGF-like domain fragment (e.g., HRG. beta. 177-244).

By "ErbB hetero-oligomer" herein is meant an oligomer that is non-covalently associated, comprising at least 2 different ErbB receptors. Such complexes may form when cells expressing 2 or more ErbB receptors are exposed to ErbB ligands, which may be separated by immunoprecipitation and analyzed by SDS-PAGE, e.g. Sliwkowski et al j.biol.chem., 269 (20): 14661-. Examples of such ErbB hetero-oligomers include the EGFR-HER2, HER2-HER3, and HER3-HER4 complexes. Furthermore, ErbB hetero-oligomers may comprise 2 or more HER2 receptors that bind to different ErbB receptors (such as HER3, HER4, or EGFR). Other proteins, such as cytokine receptor subunits (e.g., gp130), may be included in the hetero-oligomer.

The terms "ErbB 1", "epidermal growth factor receptor" and "EGFR" are used interchangeably herein to refer to the native sequence EGFR as disclosed by Carpenter et al (Ann. Rev. biochem. 56: 881-42914 (1987)), including variants thereof (e.g., deletion mutant EGFR as described by Humphrey et al (PNAS (USA) 87: 4207-4211 (1990)). ErbB1 refers to a gene that encodes the EGFR protein product. Examples of antibodies that bind to EGFR include Mab 579(ATCC CRL HB8506), Mab 455(ATCC CRLHB 8507), Mab 225(ATCC CRL 8508), Mab 528(ATCC CRL 8509) (see U.S. Pat. No. 5, 4943533) and variants thereof, such as chimeric 225(C225) and reshaped human 225(H225) (see WO 96/40210).

The terms "ErbB 2" and "HER 2" are used interchangeably and refer to the native sequence human HER2 protein, as described, for example, in Semba et al (PNAS (USA) 82: 6497-. The term erbB2 refers to the gene encoding human HER2 and neu refers to the gene encoding rat p185 neu. Preferred is HER2 native sequence human HER 2. Examples of antibodies that bind HER2 include MAbs 4D5(ATCC CRL 10463), 2C4(ATCC HB-12697), 7F3(ATCC HB-12216), and 7C2(ATCC HB12215) (see, U.S. Pat. No. 5,772,997; WO 98/77797; U.S. Pat. No. 5,840,525, which are incorporated herein by reference). Humanized anti-HER 2 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7, and huMAb4D5-8(Herceptin ), as described in table 3 in U.S. patent 5,821,337, which is incorporated herein by reference; humanised 520C9(WO 93/21319). Human anti-HER 2 antibodies are described in U.S. patent 5,772,997 and WO 97/00271.

"ErbB 3" and "HER 3" refer to receptor polypeptides, including variants thereof, as disclosed in U.S. Pat. Nos. 5183884 and 5480968 and Kraus et al (PNAS (USA) 86: 9193-9197 (1989)). Examples of antibodies that bind to HER3 are found in U.S. patent 5968511, e.g., 8B8 antibody (ATCC HB 12070) or humanized variants thereof. The terms "ErbB 4" and "HER 4" refer to receptor polypeptides disclosed in the following documents, such as european patent application 599274; plowman et al, proc.natl.acad.sci.usa, 90: 1746 — 1750 (1993); and Plowman et al, Nature, 366: 473, 475(1993), including variants thereof, e.g. the HER4 isoform as described in WO 99/19488.

An "ErbB antagonist" is any molecule that binds to an ErbB receptor and blocks ligand activation of the ErbB receptor. Such antagonists include, but are not limited to, modified ligands, ligand peptides (i.e., ligand fragments), soluble ErbB receptors, preferably anti-ErbB antibodies.

"treatment" refers to both therapeutic and prophylactic measures. Those in need of treatment are individuals already suffering from the disease as well as those in which the disease is to be prevented.

"mammal" in need of treatment refers to any animal of the mammalian family, including humans, poultry, farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cattle, etc. Preferably, the mammal is a human.

A "disease" is any condition that would benefit from treatment with an ErbB antagonist (e.g., an anti-ErbB 2 antibody), and more generally any cancer that can be treated by administration of an antibody directed against an overexpressed antigen. This includes chronic and acute diseases, or those pathological conditions that predispose a mammal to the disease. Non-limiting examples of diseases that can be treated according to the present disclosure include benign and malignant tumors; leukemia and lymphoid malignancies; neuronal, glial, astrocyte, hypothalamic and other glandular, macrophage, epithelial, interstitial and blastocoel diseases; inflammatory, angiogenic and immunological diseases.

The term "therapeutically effective amount" refers to an amount having an anti-proliferative effect. Preferably, the therapeutically effective amount elicits antibody-mediated cytotoxicity, activates complement, has apoptotic (apoptotic) activity, or is capable of inducing cell death, and preferably death of benign or malignant tumor cells, particularly cancer cells. The efficacy of treatment can be determined by conventional methods, depending on the condition to be treated. For cancer treatment, efficacy can be determined, for example, by assessing time to disease progression (TTP), survival, tumor size, or determining Response Rate (RR) (see examples below).

The terms "cancer" and "cancerous" refer to or describe the pathological state in mammals that is typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma (carcinoma), lymphoma, blastoma, sarcoma, melanoma, and leukemia. More specifically, these cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer (liver cancer), bladder cancer, hepatoma (hepatoma), breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, liver cancer (hepatoma), and various head and neck cancers.

"ErbB expressing cancer" refers to a cancer that includes cells that have ErbB proteins on their surface, such that anti-ErbB antibodies bind to the cancer.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cellular function and/or causes cellular destruction. The term is intended to include radioisotopes (e.g., I)¹³¹、I¹²⁵、Y⁹⁰And Re¹⁸⁶) Chemotherapeutic agents, toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.

A "chemotherapeutic agent" is a chemical compound used in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and Cyclophosphamide (CYTOXAN)^TM) (ii) a Alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines (aziridines) such as benzodidopa (b)enozopa), carboquone (carboquone), meturedopa (meturedopa) and uretonimine (uredopa); aziridine (ethylenimine) and melaminee include altretamine (altretamine), triimizine (triethyleneamine), triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine (trimetylomelamine); nitrogen mustards such as chlorambucil (chlorambucil), chlorambucil (chlorenaphazine), cholorophosphamide (cholorophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), mechlorethamine hydrochloride, melphalan (melphalan), neomustard (novembichin), cholestyramine phenylacetate (pherenesterodine), prednimustine (prednimustine), triamcinolone (trofosfamide), uracil mustard; nitrosoureas (nitrosureas) such as nitrosourea mustard (carmustine), chlorozotocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine), ramustine (ranimustine); antibiotics such as aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, actinomycin C (cactinomycin), calicheamicin (calicheamicin), carabacin, carcinophin (carzinophilin), chromomycin (chromomycin), actinomycin D, daunorubicin (daunorubicin), 6-diazo-5-oxo-L-norleucine, adriamycin (doxorubicin), epirubicin (epirubicin), mitomycin, mycophenolic acid, nogenin (nogalamycin), olivomycin (olivomycin), pelomomycin (polypromycin), potfifomycin, puromycin, streptonigrin (streptozotocin), tubercidin, ametocin (ubenix), stastatin (zinostatin), zorubicin (zorubicin); antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; the purine analog fludarabine (fludarabine), 6-mercaptopurine, thiamine, thioguanine; pyrimidine analogs such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine, carmofur (carmofur), cytarabine, dideoxyuridine (doxifluridine), enocitabine (enocitabine), floxuridine, 5-FU; androgens such as dimethyltestosterone (calusterone), methyl androsterone propionate(dromostanoloneproprionate), epithioandrostanol (epitiostanol), mepiquat chloride (mepiquitane), testolactone (testolactone); anti-adrenals such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); folic acid supplements such as frolicic acid; aceglucomannan lactone; (ii) an aldophosphamide glycoside; aminolevulinic acid (aminolevulinic acid); amsacrine; bestrabuucil; bisantrene; edatrexate (edatraxate); defofamine; colchicine (demecolcine); diazaquinone (diaziqutone); elfosmithine; ammonium etitanium acetate; etoglut (etoglucid); gallium nitrate; a hydroxyurea; lentinan (lentinan); lonidamine (lonidamine); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidamol (mopidamol); nifurthradine (nitracrine); pentostatin (pentostatin); phenamett; pirarubicin (pirarubicin); podophyllinic acid (podophyllinic acid); 2-ethyl hydrazide; procarbazine (procarbazine); PSK ; razoxane (rizoxane); sisofilan (sizofiran); germanium spiroamines (spirogyranium); alternarionic acid; a tri-imine quinone; 2, 2', 2 "-trichlorotriethylamine (trichlorotriethylamine); urethane (urethan); vinca amides; dacarbazine (dacarbazine); mannitol mustard; dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); a polycytidysine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa (thiotepa); taxoids, such as paclitaxel (TAXOL , Bristol-myers squibb Oncology, Princeton, NJ) and docetaxel (Taxotere, Rh  ne-Poulenc ror, antonyx, France); chlorambucil; gemcitabine (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine (vinorelbine); neomycin amide (navelbine); novantrone; teniposide (teniposide); daunorubicin; carminomycin (carminomycin); aminopterin; xeloda; ibandronate (ibandronate); CPT-11; topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); tretinoin; esperamicins; capecitabine; to be provided withAnd pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. This definition also includes anti-hormonal agents that modulate or inhibit the effects of hormones on tumors, such as anti-estrogen agents including tamoxifen (tamoxifen), raloxifene (raloxifene), the aromatase inhibitor 4(5) -imidazole, 4-hydroxytamoxifene, trioxifene (trioxifene), keoxifene, LY117018, onapristone; and antiandrogen agents such as flutamide, nilutamide; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

By "growth inhibitory agent" herein is meant a compound or composition that inhibits cell growth, particularly the growth of cancer cells that overexpress ErbB, in vivo or in vitro. Thus, the growth inhibitory agent may be a drug that significantly reduces the percentage of ErbB overexpressing cells in S phase. Examples of growth inhibitory agents include drugs that block the progression of the cell cycle (at some stage other than S phase), such as drugs that induce G1 arrest and M phase arrest. Classical M-phase blockers include vinblastines (vincristine and vinblastine), TAXOL  and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, bleomycin and the like. Those that arrest G1 also accompany (spill over) S phase arrest, for example, DNA alkylating agents like tamoxifen (tamoxifen), prednisone (prednisone), dacarbazine (dacarbazine), mechlorethamine (mechlororethamine), cisplatin, methotrexate, 5-fluorouracil, and cytarabine, among others. See, for details, The Molecular Basis of cancer; mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and antioplastic drugs" Murakami et al (WB Saunders: Philadelphia, 1995), see especially page 13. The 4D5 antibody (and functional equivalents thereof) may also be used for this purpose.

ErbB receptor tyrosine kinases

ErbB receptor tyrosine kinases are important mediators of cell growth, differentiation and survival. The receptor family includes at least four distinct members, including epidermal growth factor receptor (EGFR or ErbB1), HER2(ErbB2 or p 185)^neu) HER3(ErbB3) and HER4(ErbB4 or tyro 2).

EGFR, encoded by the erbB1 gene, is implicated in causing human malignancies. In particular, increased expression of EGFR has been observed in breast, bladder, lung, head and neck and stomach cancers as well as gliomas. Increased EGFR receptor expression is often associated with increased production of EGFR ligand-transforming growth factor alpha (TGF- α) by the same tumor cell, resulting in receptor activation by an autocrine stimulatory pathway. Baselga and Mendelsohn pharmac. ther.64: 127-154(1994). In the treatment of such malignancies, monoclonal antibodies against EGFR or its ligands TGF-alpha and EGF have been evaluated for value as therapeutics. See, e.g., Baselga and mendelsohn, supra; masui et al Cancer Research 44: 1002-1007 (1984); and Wu et al j.clin.invest.95: 1897-1905(1995).

The second member of the ErbB family, p185neu, was originally identified as the transforming gene product of neuroblastoma in chemically treated rats. The activated form of the neu proto-oncogene, derived from a point mutation (valine to glutamic acid) in the transmembrane region of the encoded protein. Amplification of the human neu homolog was observed in breast and ovarian cancers and was associated with poor prognosis (Slamon et al, Science, 235: 177-182 (1987); Slamon et al, Science, 244: 707-712 (1989); and U.S. Pat. No. 4,968,603). To date, no point mutations similar to the neu proto-oncogene have been reported in human tumors. It has been observed that overexpression of HER2 (often but not exclusively due to gene amplification) is associated with other cancers, including gastric, endometrial, salivary, lung, kidney, colon, thyroid, pancreatic and bladder cancers.

Antibodies against the murine p185neu and human HER2 protein products have been described. Drebin and colleagues have produced antibodies against the rat neu gene product-P185 neu. See, e.g., Drebin et al, Cell 41: 695-706 (1985); myers et al, meth.enzym.198: 277-290 (1991); and WO 94/22478. Drebin et al Oncogene 2: 273-277(1988) reported that the mixture of antibodies reacting with two different regions of p185neu produced a synergistic anti-tumor effect on neu-transformed NIH-3T3 cells implanted in nude mice. See also U.S. patent 5,824,311 issued on 20/10 of 1998.

Hudziak et al, mol.cell.biol9 (3): 1165-1172(1989) describe the production of a series of anti-HER 2 antibodies characterized by the use of the human breast tumor cell line SKBR 3. Relative cell proliferation was determined by crystal violet staining of monolayers 72 hours after exposure of SKBR3 cells to antibody. Using this analytical determination, the results obtained were: the maximum inhibition of cell proliferation by the antibody designated 4D5 was 56%. Other antibodies in the series of antibodies reduced cell proliferation to a lesser extent in this assay. It was further found that antibody 4D5 sensitizes HER 2-overexpressing breast tumor cell line to the cytotoxic effects of TNF- α. See also, U.S. Pat. No. 5,677,171 issued 10/14 in 1997. The anti-HER 2 antibodies discussed in Hudziak et al are further described in the following references: fendly et al Cancer Research 50: 1550 and 1558 (1990); kotts et al In Vitro 26 (3): 59A (1990); growth Regulation 1 of Sarup et al: 72-82 (1991); shepard et al j. clin. immunol.11 (3): 117-127 (1991); kumar et al mol.cell.biol.11 (2): 979-; lewis et al Cancer immunol.imother.37: 255-; pietras et al Oncogene 9: 1829-1838 (1994); vitetta et al Cancer Research 54: 5301-5309 (1994); sliwkowski et al j.biol.chem.269 (20): 14661-14665 (1994); scott et al j.biol.chem.266: 14300-5 (1991); d' souza et al proc.natl.acad.sci.91: 7202-; lewis et al Cancer Research 56: 1457-1465 (1996); and Oncogene 15 by Schaefer et al: 1385-1394(1997).

The recombinant humanized IgG1 version of the mouse anti-HER 2 antibody 4D5(rhuMAb HER2 or HERCEPTIN ; purchased from Genentech, Inc., South San Francisco) was clinically efficacious in patients with HER 2-overexpressing metastatic breast cancer who had previously received anti-cancer therapy (Baselga et al, J.Clin.Oncol.14: 737-744 (1996)). HERCEPTIN  received marketing approval from the food and drug administration at 25.9.1998 for the treatment of metastatic breast cancer patients with tumors overexpressing the HER2 protein. The current treatment protocol is the application of IHC to determine the overexpression of HER2 protein.

Other anti-HER 2 antibodies with different properties are described in the following documents: tagliabue et al int.j. cancer 47: 933 937 (1991); McKenzie et al Oncogene 4: 543 and 548 (1989); maier et al Cancer Res.51: 5361-5369 (1991); bacillus et al molecular Carcinogenesis 3: 350-362 (1990); stancovski et al PNAS (USA) 88: 8691 and 8695 (1991); bacillus et al Cancer Research 52: 2580 + 2589 (1992); xu et al int.j. cancer 53: 401-408 (1993); WO 94/00136; kasprzyk et al Cancer Research 52: 2771-2776 (1992); hancock et al Cancer Res.51: 4575-4580 (1991); shawver et al Cancer Res.54: 1367-; aretag et al Cancer Res.54: 3758-3765 (1994); harwerth et al J.biol.chem.267: 15160- > 15167 (1992); U.S. Pat. nos. 5,783,186; klaper et al Oncogene 14: 2099-2109 (1997); WO 98/77797.

Homology screening resulted in the discovery of 2 additional ErbB receptor family members; HER3 (U.S. Pat. Nos. 5,183,884 and 5,480,968, and Kraus et al PNAS (USA) 86: 9193-. Both receptors show increased expression in at least some breast cancer cell lines.

ErbB receptors are commonly found in various binding forms in cells, and heterodimerization is thought to increase the diversity of cellular responses to various ErbB ligands (ear et al Breast cancer research and Treatment 35: 115-132 (1995)). EGFR is bound by 6 different ligands: epidermal Growth Factor (EGF), transforming Growth factor alpha (TGF-. alpha.), amphiregulin (amphereregulin), heparin-binding epidermal Growth factor (HB-EGF), betacellulin (betacellulin), and epiregulin (epidermal regulin) (Groenen et al Growth factors 11: 235-257 (1994)). The family of genetic regulatory protein (heregulin) proteins produced by single gene alternative (alternative) splicing are ligands of HER3 and HER 4. The family of genetic regulatory proteins includes the alpha, beta and gamma genetic regulatory proteins (Holmes et al, Science, 256: 1205-1210 (1992); U.S. Pat. No. 5,641,869; and Schaefer et al Oncogene 15: 1385-1394 (1997)); neu Differentiation Factors (NDFs), Glial Growth Factors (GGFs); acetylcholine Receptor Inducing Activity (ARIA); and sensory and motor neuron derived factor (SMDF). Reviewed in Groenen et al Growth factors 11: 235-257 (1994); lemke, g.molec. & cell.neurosci.7: 247-: 51-85(1995). Recently, two additional ErbB ligands were identified: neuregulin-2 (NRG-2) and neuregulin-3, neuregulin-2 has been reported to bind to HER3 or HER4(Chang et al Nature 387509: 512 (1997); and Carraway et al Nature 387: 512: 516(1997)), and neuregulin-3 binds to HER4(Zhang et al PNAS (USA)94 (18: 9562-7 (1997)). HB-EGF, betacytokinin and epiregulin also bind to HER 4.

Although EGF and TGF- α do not bind HER2, EGF stimulates EGFR and HER2 to form a heterodimer, which activates EGFR and leads to phosphotransfer of HER2 in the heterodimer. Dimerization and/or transphosphorylation appear to activate HER2 tyrosine kinase. See Earp et al, supra. Similarly, when HER3 is co-expressed with HER2, an active signaling complex is formed, and antibodies against HER2 interfere with this complex (Sliwkowski et al, J.biol.chem., 269 (20): 14661-14665 (1994)). Furthermore, HER3 has an increased affinity for the genetic regulatory protein (HRG) to higher affinity levels when co-expressed with HER 2. For the HER2-HER3 protein complex, see also Levi et al, Journal of Neuroscience 15: 1329-; morrissey et al, proc.natl.acad.sci.usa 92: 1431-1435 (1995); and Lewis et al, Cancer res, 56: 1457-1465(1996). HER4, like HER3, forms an active signaling complex with HER2 (Carraway and Cantley, Cell 78: 5-8 (1994)).

Detecting gene amplification

The present invention relates to the detection of gene amplification by any technique (see, Boxer, J.Clin.Pathol.53: 19-21 (2000)). These include in situ hybridization using radioisotopes or fluorescently labeled probes (Stoler, Clin. Lab. Med.12: 215-36 (1990); Polymerase Chain Reaction (PCR); quantitative Southern blotting, and other techniques for quantifying individual genes.

The term "label" as used herein refers to a compound or composition that is directly or indirectly coupled to or fused to an agent, such as a nucleic acid probe or antibody, to facilitate detection of the agent coupled to or fused to it. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, if an enzymatic label, may be detectable to catalyze a chemical change in the substrate compound or composition. Haptens or epitopes that immunospecifically bind to the antibody may also be used as labels.

The term "fluorescently labeled nucleic acid probe" refers to a probe comprising: (1) a nucleic acid having a sequence that is capable of hybridizing to a target nucleic acid sequence; and (2) a fluorescent label. Preferably, the hybridization is specific hybridization, i.e., it can occur under highly stringent conditions.

Preparation of samples

Any tissue sample from a subject may be used. Examples of tissue samples that may be used include, but are not limited to, breast, prostate, ovary, colon, lung, endometrium, stomach, salivary gland, or pancreas. Tissue samples can be obtained by a variety of methods including, but not limited to, surgical excision, aspiration, biopsy. The tissue may be fresh or frozen. In one embodiment, the tissue sample is fixed and embedded in paraffin or the like.

Tissue samples can be fixed (i.e., preserved) using conventional Methods (see, e.g., Manual, historical stabilizing Method of The aromatic Format Institute of Pathology, 3rd edition Lee G.Luna, HT (ASCP) Editor, The Blakston Division McGraw-HillBook Company: New York; (1960); The aromatic Format Institute of Pathology Advanced laboratories in history and Pathology (1994) Ulreka V.Mikel, Editor, aromatic Format Institute of Pathology, American Registry of Washington, D.C.). One skilled in the art will appreciate that the choice of fixative will depend on whether the tissue is histologically stained or otherwise analyzed. One skilled in the art will appreciate that the length of time used for fixation depends on the size of the tissue sample and the fixative used. For example, neutral buffered formalin, Bouin's or paraformaldehyde may be used to fix tissue samples.

Typically, tissue samples are fixed, dehydrated through a series of incremental ethanol, infiltrated, and embedded with paraffin or other slicing matrix to allow sectioning of the tissue sample. Alternatively, the tissue may be sectioned and the resulting sections fixed. For example, tissue samples can be embedded and processed in paraffin by conventional methods. Examples of paraffins that may be used include, but are not limited to, Paraplast, Broloid, and tissue. Once the tissue sample is embedded, the tissue may be sectioned with a microtome or the like. For example, the slice thickness may vary from 3-5 μm. Once cut, the sections can be mounted on slides using several standard methods. Examples of slide adhesives include, but are not limited to, silane, gelatin, poly-L-lysine, and the like. For example, paraffin-embedded sections can be adhered to positively charged or poly-L-lysine coated slides.

If paraffin is used as the embedding material, the tissue sections are usually dewaxed and then rehydrated in water. Tissue sections may be dewaxed by a variety of conventional methods. For example, xylene and a decreasing series of ethanol may be used. Alternatively, commercially available non-organic dewaxers such as Hemo-De7(CMS, Houston, Texas) may be used.

In situ fluorescence hybridization (FISH)

In situ fluorescence hybridization is typically performed on cell or tissue sections that are fixed to a glass slide. In Situ Hybridization can be achieved by a variety of conventional methods (see, e.g., Leitch et al, In Situ Hybridization: A Practical Guide, Oxford BIOS Scientific Publishers, Micropscopy handbook v.27 (1994)). In one in situ hybridization method, nucleic acid sequence probes complementary to target nucleotide sequences in cells are labeled with a fluorescent dye, such as Fluorescein Isothiocyanate (FITC), which fluoresces green under excitation by an argon ion laser. Each cell containing the target nucleotide sequence will be bound to a labeled probe, and a fluorescent signal will be generated by exposing the cell to a light source having a wavelength suitable for excitation of the particular fluorescent dye used. The "target nucleotide sequence" is a specific sequence for an overexpressed tumor antigen, such as ErbB. The FISH assay may be used in conjunction with other assays including, but not limited to, morphological staining (for a series of sections or the same section; see WO 00/20641, specifically incorporated herein by reference).

Different hybridization stringencies may be utilized. The more stringent the hybridization conditions, the higher the degree of complementarity required for the probe to form and maintain a stable duplex with the target. Stringency can be enhanced by increasing temperature, decreasing concentration, or increasing formamide concentration. The addition of dextran or increasing the extraction concentration may also increase the effective concentration of labeled probe, thereby increasing the hybridization rate and increasing the limiting signal intensity. After hybridization, the solution used to wash the slides typically contains reagents similar to the components in the hybridization solution, with washing times varying from minutes to hours, depending on the desired stringency. Longer or more stringent washes generally reduce non-specific background, but risk reducing overall sensitivity.

The probe used in the FISH analysis may be an RNA or DNA oligonucleotide or polynucleotide, and it may contain not only natural nucleotides but also their analogs such as digoxigenin dCTP, biotin dCTP 7-azaguanosine, azidothymidine, inosine, or uridine. Other useful probes include peptide probes and analogs thereof, branched gene DNA, peptidomitics, Peptide Nucleic Acids (PNA), and/or antibodies.

The probe should be sufficiently complementary to the target nucleic acid sequence to allow stable and specific binding of the target nucleic acid sequence to the probe. The degree of homology required to stabilize hybridization varies with the stringency of the hybridization matrix and/or wash matrix. Fully homologous probes are preferably employed in the present invention, but one skilled in the art will recognize that probes exhibiting minor but sufficient homology may also be used in the present invention (see, e.g., Sambrook, J., et al, Molecular Cloning A Laboratory Manual, Cold spring harbor Press, (1989)).

One skilled in the art will recognize that the choice of probe depends on the nature of the target gene. Examples of amplifications include, but are not limited to, her2/neu in breast and ovarian cancer, n-myc in neuroblastoma, c-myc in small cell lung cancer. For example, to evaluate her2/neu amplification, a probe spanning a 140kb region (17q11.2-17q12) on the long arm of chromosome 17 containing the her2/neu gene can be used. Probes to the satellite sequence (D1721) in the centromere region of chromosome 17 can be used to assess aneusomy of chromosome 17 as the origin or cause of her2/neu amplification. Mixtures of these probes can be obtained, for example, from Vysis, Inc., where each probe is directly labeled with a readily distinguishable fluorescent dye, such as SPECTRUM ORANGE7 and SPECTRUM GREEN 7.

Probes can also be prepared and selected in a variety of ways including, but not limited to, mapping by in situ hybridization of the neck, grouping of somatic hybrids (panel), dot blotting of sorted chromosomes; chromosome linkage analysis; or cloned and isolated from sorted chromosome pools from human cell lines or somatic cell hybrids bearing human chromosomes, radiation treatment of the somatic cell hybrids, microdissection of chromosomal regions, or identification from Yeast Artificial Chromosomes (YACs) using PCR primers specific for unique chromosomal loci, or by other suitable means such as contiguous YAC clones. The probe may be genomic DNA, cDNA, or RNA cloned on a plasmid, phage, cosmid, YAC, Bacterial Artificial Chromosome (BAC), viral vector, or any other suitable vector. Probes may also be cloned or chemically synthesized by conventional methods. After cloning, the isolated probe nucleic acid fragment is typically inserted into a vector, such as a bacteriophage, pBR322, M13, or a vector containing the SP6 or T7 promoter, and cloned as a library in a bacterial host (see, e.g., Sambrook, supra).

The probe is preferably labeled with a fluorophore. Examples of fluorophores include, but are not limited to, rare earth metal chelates (europium chelates), Texas Red, rhodamine, fluorescein, dansyl, lissamine, umbelliferone, phycoerythrin (phytorytherin), phycocyanin, or commercially available fluorophores such as SPECTRUMORANGE7 and SPECTRUM GREEN7, and/or derivatives of one or more of the foregoing. The various probes used in the assay may be labeled with more than one different fluorescent or chromophoric color. These color differences provide a means for identifying the hybridization sites of specific probes. Furthermore, probes that are not spatially separated can be identified with a different colored light or pigment from two other colors (e.g., red + green) or a set of filters that allow only one color to pass at a time.

The probe may be directly or indirectly labeled with a fluorophore by conventional methods. Additional probes and colors can be added to refine this general approach and extend to include more genetic abnormalities or as an internal control. For example, the her2/neu gene is located on chromosome 17, and a probe (Vysis, Inc.) specific for the satellite sequence of chromosome 17 (D17Z1) can be used as an internal control to provide duplexes in regions of non-malignant cells, and/or to determine whether there is a homosomal abnormality of chromosome 17 in the her2/neu amplified region.

For FISH, the slides can be processed and analyzed by standard techniques of fluorescence microscopy (see, e.g., Ploem and Tanke, Introduction to fluorescence microscopy, Oxford University Press: New York (1987)). Briefly, each slide was observed with a microscope equipped with the corresponding excitation filter, dichroic and absorption filters (barrier filters). The filter is selected according to the excitation spectrum and the emission spectrum of the fluorescent dye used. The slide is photographed by selecting the exposure time based on the fluorescent label used, the signal intensity, and the filter selected. For FISH analysis, the physical location of the target cells determined in the morphological analysis is recorded (recall) and confirmed visually as a region suitable for FISH quantification.

To associate IHC with FISH, a computer controlled drive platform may be used that stores the location of the combined coordinates (co-ordinates). This can be used to evaluate the same region by two different analysis techniques. For example, a color image of the morphologically stained area can be captured and stored with a computer-equipped cold CCD camera. The same section is then subjected to a FISH procedure, the stored locations are recorded, and the designated area is scored to determine whether a fluorescent nuclear signal is present. A similar IHC procedure followed by FISH is also contemplated.

Typically, hundreds of cells in a tissue sample are scanned to determine the amount of specific target nucleic acid sequences, which appear as fluorescent spot counts relative to the number of cells. Deviations in the number of spots from normal (norm) alone (e.g., detection of her2/neu gene in normal cells will yield two copies, and more than two abnormalities) indicate a higher likelihood of benefit from treatment with tumor antigen-specific antibodies, such as ErbB antagonists. As described below, HER2 gene amplification was more effective in indicating the possibility of anti-HER 2 antibody therapy being effective.

Pharmaceutical preparation

Pharmaceutical formulations of the antagonists (antibodies) for use according to the invention are prepared by mixing the antibody of the desired purity with an optional pharmaceutical carrier, excipient or stabilizer (Remington's pharmaceutical Sciences 16 th edition, Osol, A. eds. (1980)), and then stored as a lyophilizate or as an aqueous solution. Carriers, excipients, stabilizers which may be used are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (for example octadecyl dimethyl benzyl ammonium chloride; hexane diamine chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino groupAcids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and/or nonionic surfactants such as tweens^TM，PLURONICS^TMOr polyethylene glycol (PEG). Preferred lyophilized anti-ErbB 2 antibody dosage forms are described in WO97/04801, which is incorporated herein by reference.

The formulations may also comprise more than one active compound, depending on the particular situation to be treated, preferably those having complementary activities without adversely affecting each other. For example, it may be desirable to further provide antibodies that bind to EGFR, ErbB2, ErbB3, ErbB4, or Vascular Endothelial Growth Factor (VEGF), or antibodies that bind to different epitopes on ErbB, in one formulation. Alternatively (or additionally), the composition may further comprise a cytotoxic agent, a chemotherapeutic agent, a cytokine, a growth inhibitory agent, and/or a cardioprotective agent. These molecules are suitably present in a combination in a total amount effective for the desired purpose.

The active ingredient may also be contained in microcapsules prepared by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly (methylmethacylate) microcapsules, respectively, in drug delivery systems of colloidal nature (such as liposomes, albumin microbeads, microemulsions, nanoparticles and nanocapsules) or macroemulsions (macroemulsions). These techniques are described in Lee's pharmaceutical, Osol, 16 th edition, eds (1980).

Formulations for in vivo administration are preferably sterile, and must be sterile for use in humans. This can be easily achieved by filtration through sterile filtration membranes.

Controlled release formulations may also be prepared. Suitable examples of controlled release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of controlled release formulations include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate)Esters) or poly (vinyl alcohol), polylactide (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid with gamma ethyl-L-glutamate, non-degradable ethylene ethyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRONDEPOT^TM(injectable microspheres consisting of lactic-glycolic acid copolymer and leuprolide acetate), and poly D- (-) -3-hydroxybutyric acid. Although polymers such as ethylene-ethyl acetate and lactic acid-glycolic acid release the molecule for over 100 days, some hydrogels release the protein for a shorter period of time. When encapsulated antibodies stay in the body for a long time, they are denatured or aggregated by exposure to a humid environment at 37 ℃, resulting in loss of biological activity, and immunogenicity may be changed. Rational strategies for stabilization can be devised based on the relevant mechanism. For example, if the mechanism of aggregation is found to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling humidity, employing appropriate additives, and developing specific polymer matrix compositions.

Treatment with anti-ErbB antagonists

According to the invention, anti-ErbB antibodies or other antagonists may be used to treat a variety of conditions characterized by overexpression and/or activation of ErbB receptors in patients in which amplified ErbB genes are found. Examples of conditions include benign or malignant tumors (e.g., renal (renal) carcinoma, liver cancer, kidney (kidney) carcinoma, bladder cancer, breast cancer, stomach cancer, ovarian cancer, colorectal cancer, prostate cancer, pancreatic cancer, lung cancer, vulvar cancer, thyroid cancer, liver cancer (hepatoma), sarcomas, glioblastomas and various types of head and neck cancers); leukemia and lymphoid malignancies; other diseases, such as disorders of nerve cells, glial cells, astrocytes, hypothalamus, glands, macrophages, epithelium, stroma and blastocoel, inflammatory, angiogenic and immunological disorders.

The antibodies, chemotherapeutics and any other active agents of the invention can be administered to a patient according to known methods, for example, intravenously via rapid infusion or by continuous infusion over a period of time, intramuscularly, intraperitoneally, intracerobrospinally, subcutaneously, intraarticularly, intrasynovially, intrathecally, orally, topically or by inhalation. Intravenous or subcutaneous administration of the antibody is preferred.

In one embodiment, the treatment of the invention involves the administration of an anti-ErbB antibody in combination with a chemotherapeutic agent (e.g., taxoid). The present invention relates to the administration of mixtures of different chemotherapeutic agents. Co-administration includes co-administration (using separate formulations or a single pharmaceutical formulation) and sequential administration in any order, where it is preferred that there is a period of time for both (or all) active agents to exert their biological activities simultaneously. The formulation and dosage regimen of these chemotherapeutic agents can be used according to the manufacturer's instructions or determined according to the practical experience of a skilled physician. Formulations and administration protocols for such chemotherapies are also described in chemotherapy ed, m.c. perry, Williams and Wilkins, Baltimore, MD (1992). The chemotherapeutic agent may be administered before, after, or simultaneously with the administration of the antibody. The antibody may be administered in combination with an anti-estrogenic compound (e.g. tamoxifen) or an anti-progestin (e.g. onapristone) in known doses of these molecules (see EP 616812).

In addition to the above treatment regimens, the patient may receive surgical resection of cancer cells (tumor resection) and/or radiation therapy.

For prophylactic or therapeutic treatment of a disease, the appropriate dosage of an antagonist (e.g., an antibody) will depend on the type of disease being treated (as described above), the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attendant physician. The antibody is suitably administered to the patient at once or over a series of treatments.

Depending on the type and severity of the condition being treated, the initial candidate dose of antibody administered to the patient is about 1. mu.g/kg to 15mg/kg (e.g., 0.1 to 20mg/kg), whether administered, for example, by one or more divided administrations, or by continuous infusion. Typical daily dosages will be in the range of about 1. mu.g/kg to 100mg/kg or more, depending on the factors mentioned above. With respect to repeated administration for several days or longer, the treatment should be continued as appropriate until the desired suppression of disease symptoms occurs. But other dosage regimens may be applied. The progress of the treatment is readily monitored by routine techniques and experimentation.

A medicine bag; article of manufacture

Related aspects of the invention provide articles of manufacture containing materials useful in the treatment of the above-mentioned disorders. The article comprises a container, optionally with a label and a package insert. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be made of various materials such as glass or plastic. The container may contain a composition effective for treating the condition and preferably has a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a therapeutic antibody against a tumor antigen or an ErbB antagonist, such as an anti-ErbB antibody. The label on or attached to the container indicates that the composition can be used to treat a given disease. Furthermore, the article of manufacture may comprise a second container containing a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution and dextrose solution. The second buffer may be used to reconstitute the active agent in lyophilized or dry powder form, or to dilute a concentrated preparation of the active agent. It may also include other substances that are commercially desirable or advantageous to the user, including other buffers, diluents, filters, needles and syringes.

In addition, the article of manufacture comprises a package insert or insert that can direct how to use in patients who have been tested for amplification of the erbB gene by FISH. Such patients may be those who have been excluded from ErbB antagonist therapy by IHC testing, e.g., patients scored 0 or 1+ with an anti-HER 2 antibody.

Preservation of biological materials

The following hybridoma cell lines were deposited with the American type culture Collection, 12301 ParklawnDrive, Rockville, Md., USA (ATCC):

antibody nomenclature ATCC accession number

7C2 ATCC HB-122151996 year 10 months 17 days

7F3 ATCC HB-122161996 year 10 month 17 day

4D5 ATCCCRL 104631990 years 5 months and 24 days

More details of the invention are illustrated with reference to the following non-limiting examples.

Example 1: clinical Trial (Trial) analysis of the identity of (CTA) with in situ fluorescence hybridization (FISH) in the Herceptin  Critical Trial (Pivotal Trial)

Overexpression of HER2 was able to reach 2+ or 3+ levels as measured by Immunohistochemistry (IHC), a prerequisite for inclusion in the critical Herceptin  metastatic breast cancer test. Clinical Test Assays (CTA) included two IHC assays performed with monoclonal antibody 4D5 (after digestion of formalin-fixed samples with protease) or CB 11 (after heat treatment of formalin-fixed samples). A subject is eligible if it scores 2+ or 3+ in any of the assays. If both tests are performed, the final score is higher than both results.

The identity of CTA with another IHC, HercepTest (HT), was 79%. This is the basis for FDA approval of HT for the aid in the selection of patients receiving Herceptin therapy.

This example describes a similar consistency study using clinical material submitted for screening for Herceptin  -critical experiments comparing the amplification of the her2/neu gene as determined by PathVysis FISH analysis. In these key experiments, 5998 subjects were screened for HER2 expression; 1915 cases (32%) were positive for CTA, and 4083 cases (68%) were negative. 623 specimens (1: 1 positive: negative) were randomly selected for this analysis, 317 cases were CTA + and 306 cases were CTA-. The specimen is not freshly excised from the tissue mass. They are 4-6 μ sections on glass slides that have been stored for 2-4 years. Amplification of her2/neu was analyzed for each section using the protocol explicitly shown in the package insert of PathVysion. Amplification is defined as a signal ratio greater than or equal to 2. The results are shown in Table 1.

TABLE 1 FISH/CTA identity

CTA
			0 1+ 2+ 3+
-FISH+	207 28 67 217 2 21 176
				4％ 7％ 24％ 89％	529

FISH + ═ HER 2: CEP17 signal ratio is more than or equal to 2, consistency is 82% (79-85%)

Of a total of 623 specimens tested, 529 cases obtained FISH signals. There were 19.9% of CTA-samples and 10.4% of CTA + samples that failed the analysis. The 0, 1+, 2, and 3+ scores were amplified by 4.2%, 6.7%, 23.9%, and 89.3%, respectively. The identity of the samples was 81.3%, similar to a 79% CTA/HT identity. Single copy overexpression was 31%, predominant in the 2+ score group. Amplification was rare in the CTA-group (4.6%). The higher test failure rate of the CTA-group may be due to factors unrelated to the test, such as tissue fixation. This may lead to false negative results for IHC.

These data are likely to suggest: the her2/neu amplification status has unexpected predictive value over Herceptest for identifying patients more likely to benefit from Herceptin treatment. Only 24% of 2+ patients are FISH +, a finding suggesting: this subgroup is less predictable for therapeutic outcome by IHC screening alone. Identification of FISH + patients in subgroups scoring 1+ and 0, it was possible to identify those patients who were not able to receive Herceptin  treatment according to IHC criteria, but who are likely to benefit from Herceptin  treatment. Direct analysis of Herceptin  benefit based on FISH scores (compared to IHC scores) is shown in example 2.

Example 2: FISH/clinical outcome study

This example relates the results of three Herceptin  tests to FISH status. In this study, 805 subjects were randomly selected from all three trials. Of these 167, the slice was missing. Another 78 failed analyses (9.7%). Thus, 540 sections fixed in formalin and stored for 2.5-4.5 years constitute all samples of the study. There was no imbalance in the demographic or prognostic indicators in these samples. The different treatment groups had results.

The correlation of FISH status with response was assessed for those patients receiving Herceptin as second or third line therapy. These data in table 2 are for patients scored as 2+ and 3+ (by CTA).

TABLE 2 FISH/response to Herceptin  Single agent, second or third line therapy, 2+/3+ Mixed group

	FISH+	FISH-
			Answering	21	0
No answer	84	37
			Response rate	20％	0％

(12.5-27.5％) (0.7％)

N＝142

The 20% response rate of FISH + subjects unexpectedly exceeded the 15% response rate of 2+ and 3+ patients in this study, as well as the 14% response rate of patients selected by CTA and 2+ or 3+ immunohistochemistry during the critical trial. Although FISH is very correlated with IHC to the same extent as in another IHC trial, Hercep Test, shown in example 1, it is surprisingly more advantageous to identify patients who are more likely to benefit from Herceptin treatment.

These data were divided into 3+ and 2+ patient groups and the same 20% response rate was observed for FISH + patients (tables 3 and 4).

TABLE 3 FISH/response to Single agent Herceptin , second or third line therapy, subgroup 3+

	FISH+	FISH-
			Answering	18	0
No answer	72	17
			Response rate	20％	0％

(12-28％) (0-14％)

N＝107

TABLE 4 FISH/response to Single agent Herceptin , second or third line therapy, subgroup 2+

	FISH+	FISH-
			Answering	3	0
No answer	12	20
			Response rate	20％	0％

(1-40％) (0-14％)

N＝35

In the 3+ subgroup, the FISH + response rate (20%) was very close but still exceeded 17% of the response rate of the 3+ subjects. The 2+ subgroup differs significantly, with only 9% response rate, whereas subjects selected by FISH + were 20%. These data indicate that FISH + status (her2 gene amplification) greatly increases the likelihood of responding to Herceptin .

Data on patient response to Herceptin as first line treatment was also evaluated (table 5).

TABLE 5 FISH/response to Herceptin  Single agent, first line therapy, 2+/3+ Mixed group

	FISH+	FISH-
			Answering	17	1
No answer	24	20
			Response rate	41％	20％

(26-56％) (0-14％)

N＝62

The response rate of 41% in FISH + subjects was significantly higher than that of 27% in 3+, 2+ subjects.

The unexpectedly increased likelihood of a favorable response based on FISH analysis extended to a response to chemotherapy plus Herceptin , see table 6. FISH + subjects showed much stronger responses to chemotherapy and Herceptin (54%) than to FISH (41%). Tables 7-9 contain more detailed data, which are scattered by different chemotherapeutic agents (adrinomycin and cyclophosphamide AC; Paditaxol, P) and different endpoints (response rate, time to progression, and survival) when Herceptin  is treated in combination with chemotherapy.

TABLE 6 FISH/response to Herceptin  Single agent, first line therapy, 2+/3+ Mixed group

	C alone	C+H
			FISH-	39％(26-52％)	41％(27-55％)
FISH+	27％(19-35％)	54％(45-63％)

N＝336

TABLE 7 response rates of newly identified populations

		H+Ac(n＝143)	AC(n＝138)	H+P(n＝92)	P(n＝96)	H+CT(n＝235)	CT(n＝234)
								2+/3+	469	56*	42	41*	17	50*	32
3+	349	60*	42	49*	17	56*	31
								FISH+	240	58*	40	49*	14	54*	27

^*p＜0.05

TABLE 8-time of progression (month) for newly defined population

		H+Ac(n＝143)	AC(n＝138)	H+P(n＝92)	P(n＝96)	H+CT(n＝235)	CT(n＝234)
								2+/3+	469	7.8*	6.1	6.9*	2.7	7.4*	4.6
3+	349	8.1*	6.0	7.1*	3.0	7.8*	4.6
								FISH+	240	7.8*	6.2	7.0*	3.2	7.3*	4.6

^*p＜0.05

TABLE 9 survival of newly defined population (month)

		H+Ac(n＝143)	AC(n＝138)	H+P(n＝92)	P(n＝96)	H+CT(n＝235)	CT(n＝234)
								2+/3+	469	27	21	22	18	25*	20
3+	349	31*	21	25	18	29*	20
								FISH+	240	29*	20	25*	14	27*	18

^*p＜0.05

These data agree that FISH + analysis, although closely related to IHC, more accurately indicates the likelihood of success of treatment with Herceptin. In all cases, the FISH + selection gave a response rate of about 1/3 (30%) higher than the 2+/3+ IHC-selection. In 2+ patients, the FISH status provides a more efficient tool for selecting patients. FISH status also identifies patients who are excluded from treatment due to identification by IHC as either a 0 or 1+ status.

These findings generally broaden the scope of ErbB receptor antagonist-based cancer therapy and anti-tumor antigen cancer therapy. Thus, the likelihood of efficacy is increased when an erbB antagonist, e.g. an anti-erbB receptor antibody (e.g. Herceptin ) is administered to a patient judged positive for erbB gene amplification, e.g. by FISH. From these data, this is true when Herceptin  is used.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

It is also to be understood that the values are approximate and are for illustration.

Patents, patent applications, publications, product specifications, and embodiments (protocols) are cited throughout this application in their entireties, all of which are incorporated herein by reference.

Claims (23)

1. A method of increasing the likelihood of effectiveness of an ErbB antagonist cancer treatment comprising administering a cancer treating dose of an ErbB antagonist to subjects who have been found to have ErbB gene amplification in tumor cells of a tissue sample thereof.

2. The method of claim 1 wherein said ErbB is HER2 protein.

3. The method of claim 2, wherein the cancer is breast cancer.

4. The method of claim 3, wherein the subject, upon immunohistochemical analysis of a sample of the formaldehyde-fixed tissue, is scored as 0 or 1 +.

5. The method of claim 1, wherein the ErbB antagonist is an anti-ErbB antibody.

6. The method of claim 5, wherein said ErbB is HER2 and said antibody is recombinant human monoclonal antibody (rhuMAb)4D 5.

7. The method of claim 1 wherein amplification of the erbB gene is detected by detecting fluorescence of a fluorescently labeled nucleic acid probe that hybridizes to the gene.

8. The method of claim 7 wherein said erbB gene is her2 gene.

9. The method of claim 1, further comprising administering a cancer treating dose of a chemotherapeutic agent.

10. The method of claim 9 wherein said ErbB is HER2 and said chemotherapeutic agent is paclitaxel.

11. The method of claim 1, wherein the likelihood of effectiveness is increased by about 30%.

12. A method of increasing the likelihood that an anti-HER 2 antibody will be effective in treating cancer, the method comprising administering a cancer treating dose of an anti-HER 2 antibody to subjects who have been found to have had the HER2 gene amplified in tumor cells of a tissue sample thereof.

13. The method of claim 12, wherein the subject, upon immunohistochemical analysis of a sample of the formaldehyde-fixed tissue, is scored as 0 or 1 +.

14. The method of claim 12, further comprising administering a cancer treating dose of paclitaxel.

15. A medication pack comprising:

(a) a container containing an ErbB antagonist for treating cancer; and

(b) instructions for administering an erbB antagonist to those patients whose tumor cells in the tissue sample have amplified the erbB gene.

16. The pharmaceutical package of claim 15, wherein said ErbB antagonist is an antibody.

17. The pharmaceutical package of claim 16, wherein the antibody is an anti-HER 2 antibody.

18. The pharmaceutical pack of claim 17, wherein the anti-HER 2 antibody is rhuMAb 4D5(Herceptin )

19. The pharmaceutical package of claim 15, wherein the instructions further comprise directions for administering a chemotherapeutic agent in combination with the ErbB antagonist.

20. The pharmaceutical package of claim 19, wherein the chemotherapeutic drug is paclitaxel.

21. A method of identifying a patient who has been treated to respond preferentially to an ErbB antagonist for the treatment of cancer, the method comprising detecting amplification of the ErbB gene in tumour cells of a tissue sample from the patient.

22. The method of claim 21, wherein the subject, upon immunohistochemical analysis of a sample of the formaldehyde-fixed tissue, is scored as 0 or 1 +.

23. The method of claim 21 wherein said erbB is her 2.